Sheath Flow Methods for Fabricating Structures

ABSTRACT

A sheath flow system having a channel with at least one fluid transporting structure located in the top and bottom surfaces situated so as to transport the sheath fluid laterally across the channel to provide sheath fluid fully surrounding the core solution. At the point of introduction into the channel, the sheath fluid and core solutions flow side by side within the channel or the core solution may be bounded on either side by the sheath fluid. The system is functional over a broad channel size range and with liquids of high or low viscosity. The design can be readily incorporated into microfluidic chips without the need for special manufacturing protocols. Uses include extruding materials and/or fabricating structures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §§120 and 121 of U.S.application Ser. No. 14/725,769 filed on May 29, 2015 which claimsbenefit under 35 U.S.C. §§120 and 121 of U.S. application Ser. No.12/987,251 filed on Jan. 10, 2011 which claims the benefit under 35U.S.C. §§120 and 121 of U.S. application Ser. No. 11/423,225 filed onJun. 9, 2006, which claims the benefit under 35 USC §119(e)) of U.S.Application No. 60/690,057 filed on Jun. 9, 2005. The entirety of eachof these applications is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Sheath flow is a widely used technique for a variety of applications,including but not limited to particle counting, flow cytometry,waveguiding, and fluid control. Sheath flow involves surrounding acentral flow stream (the core) with a surrounding stream (the sheath).In particle counting and flow cytometry applications, the sheathprevents particles in the core from coming into contact with the wallsof the channel, thus preventing adhesion and clogging. The sheath alsoserves to focus the particles or molecules into the center of thechannel, allowing for easy counting or measurement through optical orother means. Sheath flow can also be used with fluids of differentrefractive index to create a waveguide in the core or sheath stream inorder to measure transfer of analytes from one stream to the other or tocontrol the rate of interaction between molecules in one or both streamsfor carefully controlled chemistry or analysis.

Previous designs have created sheath flow through an annulararrangement. A small nozzle was positioned inside a larger tube. Thecore solution was pumped through the nozzle and the sheath solution waspumped through the larger tube. This configuration required carefulalignment of the two tubes and did not easily lend itself tominiaturization. Since the diameter of the nozzle was fixed, therelative sizes of the core stream and sheath solution were relativelyconstant within a set range.

Other devices provide sheath flow on a chip, but the flow typicallyoperates only in two dimensions. The core stream in these devices isbordered on either side by the sheath streams, however the core is notsheathed top and bottom. The complexity of the support plumbing forthese devices is increased, as the number of flow streams is increasedfrom two to three as compared to the annular arrangement design. It ispossible to sheath the stream on the top and bottom of the core streamin these systems by adding two additional inlet ports in the top andbottom of the channel. However, this greatly increases the manufacturingcomplexity of the device. Micromachining technologies are inherentlytwo-dimensional. Three-dimensional channel paths can be created bystacking several two dimensional designs on top of one another, but thisadds to the complexity and difficulty of the manufacturing process.Creating a fully sheathed flow in this way could require at leastseveral individual levels, which must be independently produced and thencarefully aligned. In addition, use of the device could require multiplepumps to provide solutions to all the inlets.

Flow cytometry is a common technique used to count and evaluate cellsand other particles in suspension. In traditional flow cytometers, thesample solution exits a small tube into the center of a larger tube,carrying clean solution. The larger tube is then constricted so thatboth streams are reduced in diameter and accelerate. The sample streamis reduced in diameter to roughly the size of the cells to be analyzed.This forces the cells to travel in single file, along a fixed and highlyprecise trajectory within the flow channel. Because the cells arepositioned so reproducibly, high numerical aperture optics can beprecisely aligned to interrogate them. Alternatively, electronicmethods, such as capacitance or impedance changes, could be used forinterrogation. Also, because the cells are all following the same pathdown the channel, they all have the same velocity. This allows theduration and intensity of signals to be correlated with individual cellsand particles.

Because of the success of bench-top cytometers, there have been severalattempts to create a miniaturized flow cytometer. The laminar flow foundin most microfluidic systems makes them at least theoretically wellsuited to flow cytometry. In practice, however, emulating the annulardesign of the traditional cytometers is a difficult fabrication problem.

Some flow cytometers operate by simply filling the whole channel withthe sample stream. Optical detection can be problematic in these systemsbecause the cells are evenly distributed in the channel. Reducing thedimensions of the channel makes it easier to focus the optics tightlyonto the cells, but also increases the risk of clogging. Light scatteroff the walls of the channel is also a problem with these systems.Another flow cytometer operates by confining the flow top and bottombetween two hydrophobic PDMS layers, and on the sides by air. A varietyof factors effect the size of the “channel,” including the hydrostaticpressure and surface tension of the fluid. This system also suffers fromthe light scattering issues of the previous designs. In addition anycontamination of the PDMS surface will change the containment of thesolution and may ultimately cause it to fail.

Another flow cytometer system approximates an annular design by focusingthe sample stream in one dimension. The sample stream was passed throughone arm of a cross intersection while sheath streams are introducedthrough the two perpendicular arms to laterally constrict the samplestream to the center of the channel. The sample is only confined on thesides, therefore the cells come in contact with the top and bottom ofthe channel, creating the risk of fouling, and often necessitating theaddition of a dynamic coating such as bovine serum albumen,hydroxylpropylmethyl cellulose, or covalent coatings such astrichlorohexadecylsilane. Also, the fact that the cells are distributedvertically means that the optics must have a relatively low numericalaperture, which decreases the amount of light that can be gathered froma single cell and reduces the spatial resolution of the measurements.

Other flow cytometer systems attempt to sheath the sample stream bothhorizontally and vertically, typically by adding an additional twochannels to sheath the stream vertically as well as horizontally. Fromthe standpoint of cytometry, this is a far better situation, because thesample is now completely isolated from the channel walls, and theposition of the particles to be analyzed is fixed. Unfortunately, theaddition of another set of sheath inputs brings the total number tofour. Their relative flow rates must be carefully controlled or theposition of the sample stream will drift and the particles will nolonger pass through the aligned interrogation region. The best way toensure even distribution of flow among all the sheath channels is tohave a separate pump supplying each stream, but this substantiallyincreases the expense and complexity of the supporting fluidics.

Therefore there is a need in the art for a method and device ofproviding a sheath flow that fully surrounds the core, can be varied insize, and is easy to manufacture and use for a wide variety ofapplications.

BRIEF SUMMARY OF THE INVENTION

Streams in microfluidic systems with low Reynolds numbers operate in thelaminar flow regime, e.g. there is no turbulent mixing or transport ofsolutes between the streams other than occurs through diffusion.Provided is a sheath flow method and device comprising a channel havingat least one groove in the top and bottom of the channel. Core fluid isintroduced into one side of the channel, while sheath fluid isintroduced on the other side. The grooves or ridges cause sheath fluidto flow from one side of the channel entirely around the core fluid tothe other side of the channel. Thus the core fluid introduced into oneside is entirely encircled by sheath fluid introduced into the otherside.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view of one example of a sheath flow device;

FIG. 2 is a view of one example of a sheath flow device;

FIGS. 3( a) through 3(f) show the cross-sections resulting from a sheathflow device having 1 pair of grooves through 6 pairs of grooves,respectively;

FIG. 4 is a view of one example of a sheath flow device;

FIG. 5 is a view of one example of a sheath flow device;

FIG. 6 is a representative cross section of sheathed flow;

FIG. 7 is a representative cross section of sheathed flow;

FIG. 8 is a representative cross section of sheathed flow;

FIG. 9 is a representative cross section of sheathed flow;

FIG. 10 is a representative cross section of unsheathed flow;

FIGS. 11 a-c show systems in which the relative flow rates of the corestream 28 and sheath stream 26 are adjusted so that the core diameter isvery small compared to the sheath;

FIG. 12 is a liquid waveguide device;

FIG. 13 is a representation of waveguided light though a liquidwaveguide;

FIG. 14 is a near field microscope;

FIG. 15 is a flow cytometer device;

FIGS. 16( a)-16(e) show a series of traces of the light scatterresulting from the series in order of increasing concentration;

FIG. 17 is a view of one example of a sheath flow device;

FIG. 18 is a tube within a tube made by the sheath flow device; FIG. 19a is view of one example of a sheath flow device showing fluidtransporting structure across the top surface and a second fluidtransporting structure across the bottom surface; and FIG. 19 b is viewof one example of a sheath flow device showing fluid transportingstructure across the top surface and a second fluid transportingstructure across the bottom surface.

DETAILED DESCRIPTION OF THE INVENTION

In the present device and method, the core stream and one or more sheathstreams are introduced into a single channel. One or more fluidtransporting structures located at the top and bottom of the channeldirect the sheath fluid around the core stream, separating the corestream from the walls of the channel. Once the position of the corestream is established in the interior of the channel, it remains in thatposition due to laminar flow.

FIG. 1 shows a top view of one example of a sheath flow device. A sheathstream inlet 10, and a core stream inlet 12, allow a sheath stream and acore stream to be introduced into a channel 14. One design provides fora at a ‘T’ intersection at the proximal end 16 of the channel 14. Thesheath stream and the core stream flow down the channel side-by-sidetowards the distal end of the channel 18 where an outlet 20 is present.At least one fluid transporting structure 22 such as a groove or a ridgeis located in the channel 14 between the inlets 10, 12 and the outlet20. The fluid transporting structure 22 transports the sheath streamacross the top and bottom of the channel 14 to completely surround thecore stream. The fluid transporting structure 22 crosses the channel 14at an angle 30.

The device can be readily fabricated using a variety of techniques,including molding, milling, laser ablation, soft lithography techniquesand other fabrication techniques known to those skilled in the art. Anymaterial that can be machined or molded into the appropriate shapes canbe used. The current techniques used in the mass production ofmicrofluidic components can be easily adapted to the production of thissheath flow design.

The exact shape of the channel is not critical. For example, FIG. 2shows a channel 14 with a constriction at the location of the grooves22. The constricted device showed similar behavior to devices withoutthe constriction. The size of the channel can be varied within a broadrange of size scales. The size of the channel is limited at the lowerend by diffusion. When the width or diameter of the channel reaches thediffusional distance of the molecules or particles of interest, anyattempts to confine them to a specific region of the channel will bethwarted.

The upper limit for the channel width is set by the Reynolds number ofthe system. The device shown in FIG. 1 has been shown to function atReynolds numbers up to and including 200. This means that the device canbe fabricated into larger sizes using slower velocities or higherviscosity fluids. Sheath flow devices have been fabricated for use withhigh viscosity fluids that are 3 mm in width that have Reynolds numbersof 0.0008, so the actual channel diameter can be significantly widerthan that with the use of appropriate fluids. The device will operate atReynolds numbers up to those at which turbulence is initiated.

The channel has at least two inlets at or near its proximal end. Theinlets are used to introduce a sheath stream and a core stream into thechannel. The size and exact location of the inlets are not important,provided that the fluid transporting structure in the channel is locateddownstream from the inlets.

The at least one fluid transporting structure is typically a groove or aridge located inside the channel. The structure transports the sheathstream laterally across the channel and around the core stream,separating the core stream from the walls of the channel. Once theposition of the core stream is established in the interior of thechannel, it remains in that position due to laminar flow. The angle ofthe fluid transporting structure across the channel is not critical tothe design. FIG. 1 shows a device having a fluid transporting structure22 that has an angle 30 that is about 45° relative to the channel;however, other oblique angles will work as well.

The number and depth of the fluid transporting structures are designparameters that also can be adjusted to suit particular applications. Asingle structure located on the top and bottom of the channel willprovide for a full sheath around the core stream. The grooves do nothave to be aligned in order for the device and method to operate.Increasing the number of fluid transporting structures provides controlover the lateral position of the core within the channel. Increasing thesize of the fluid transporting structures correlates with a moreeffective transport of the sheath stream across the channel. Preferably,the fluid transporting structures penetrate the wall of the channel onthe downstream end. FIG. 1 shows the fluid transporting structures 22penetrating the wall of the channel 14 on the downstream end. Thispenetration increases the effectiveness of the fluid transport to betterencase the core stream in the sheath stream. Sheathing will occur,however, even if the fluid transporting structure does not penetrate thechannel wall. FIGS. 19 a and 19 b show a two embodiments of the presentsheath flow device having a first fluid transporting structure 22located across a top surface 60 of a channel and a second fluidtransporting structure 22 located across a bottom surface 62 of achannel.

Example: The number of grooves can be used to control the position ofthe core within the channel. FIGS. 3( a) through 3(f) show thecross-sections resulting from a sheath flow device having 1 pair ofgrooves through 6 pairs of grooves, respectively. One pair of grooves issufficient to completely surround the core stream 28 with sheath stream26. FIG. 3( a) illustrates the top surface 60 of the channel and thebottom surface 62 of the channel. Subsequent pairs carried more sheathfluid to the right, causing the core to be shifted leftward. Having fourpairs of grooves appears to be sufficient to place the core roughly inthe center of the channel. Depending on the relative flow rates of thetwo fluids, the core can be made as small as 1% of the total channelcross-section. It is also possible to make the core quite large withoutlosing the sheathing effect.

The fluid transporting structures may also be used in a crossconfiguration when sheath solution is provided from both sides by athird inlet. FIG. 4 shows a channel, 14, having a first sheath streaminlet 10 and a second sheath stream inlet 24. The core stream inlet 12is located between the first and second sheath stream inlets. A firstgroove 22 located in the top of the channel moves sheath stream from theleft of the channel across the top. An opposing groove 22 located at thebottom the channel in a cross configuration with the first groove movessolution from the right of the channel across the bottom. This designhas the advantage that the centroid of the core remains stationary, evenwhen the relative flow rate of the core solution is varied.Additionally, the first and second sheath stream inlets allow differingsheathing materials to be introduced into the channel.

Further, the fluid transporting structures located on the top and bottomof the channel may be configured in a shape that crosses the channelhaving a central area that is distal to its ends, as show in FIG. 5. Thefluid transporting structure 22 of FIG. 4 is shown as a “v” shape,however, any shape having a central area that is located distally in thechannel to its ends would work, such as a semi-circle. FIG. 4 shows achannel, 14, having a first sheath stream inlet 10 and a second sheathstream inlet 24. The core stream inlet 12 is located between the firstand second sheath stream inlets. Fluid transporting structures 22located in the top of the channel moves sheath stream across the corestream to sheathe the core stream.

Example: A microfluidic chip was made using a Techno-isel CNC millingrouter (Techno Inc., New Hyde Park, N.Y.) in poly(methylmethacrylate)(PMMA) (Plexiglas G, Atofina Chemical Inc., Philadelphia, Pa.) via amethod described by Howell, et al, Lab on a Chip 2005, 5, 524-530,Howell, et al, Lab on a Chip 2004, 4, 663-669, and Mott, et al, Lab on aChip 2006, 6, 540-549, all incorporated in full herein by reference. Themain channel was 3.18 mm wide by 1.02 mm deep. The grooves were 0.794 mmwide by 0.51 mm deep, and placed in pairs on both the top and bottom ofthe channel. A 70% fructose solution was used as core and the sheathsolutions to ensure that the flow within the channel stayed in theStokes regime. The sheath stream was labeled with fluorescent dye(Rhodamine WT, Bright Dyes, Miamisburg, Ohio). Channel cross-sectionsdownstream of the grooves were obtained via a method describedpreviously by Howell, P. B. et al, Lab on a Chip 2005, 5, 524-530 andMott, et al, Lab on a Chip 2006, 6, 540-549, both incorporated in fullherein by reference.

The relative flow rate of the two streams can be widely varied withoutcompromising the integrity of the sheath. FIG. 6 demonstrates acore-to-sheath ratio of 4:1. While the volumetric flow rate of thesheath stream 26 constitutes just 20% of the channel, it stillcompletely surrounds the core stream 28. FIG. 7 demonstrates that acore-to-sheath ratio of 1:4. While the core stream 28 has been reducedto 20% of the net flow compared to the sheath stream 26, it is stillclearly defined. For the specific device and method used in the example,a stable, fully enveloped sheath flow for Reynolds numbers of up toapproximately 200 was generated before the limits of the pump werereached.

FIG. 8 shows a typical cross-section of the channel before sheathing.Sheath stream 26 and core stream 28 are side by side in the channel.FIG. 9 shows the sheath stream 26 surrounding the core stream afterpassing the fluid transporting structures, not shown. Fluorescent dyecan be added to either the sheath stream or the core stream to providecontrast. Unlike other sheath flow systems, this device has also beenshown to be reversible. It is possible to unsheathe a sheathed flow torecapture both the core and the sheath with high efficiency. Unsheathingis achieved by providing a second fluid transporting structure locatedproximally in the channel from the first fluid transporting structure.The second fluid transporting structure is arranged with a reversal ofdirection as compared to the first fluid transporting structure. Thesecond fluid transporting structure does not have to be arranged to bethe exact reverse of the first fluid transporting structure, however,the orientation is in the opposite direction from the first. The abilityto unsheathe a sheathed flow can be useful in systems where the sheathsolution is in limited supply and the capability of recycling the flowis advantageous, such as continuous monitoring on a space station orother enclosed environment. It would also be useful where the solute orparticles in the core solution were very precious and recapture isimportant. FIG. 10 shows the sheath stream 26 and the core stream 28after unsheathing.

The diameters of the sheath and core can vary widely depending on theintended use of the device. FIGS. 8-10 show cross sections of a sheathflow system where the flow rate of the sheath stream is approximatelythe same as that of the core fluid and the sheath and the core havesimilar cross sectional areas. FIGS. 11 a-c show systems in which therelative flow rates of the core stream 28 and sheath stream 26 areadjusted so that the core diameter is very small compared to the sheath(<16 micron core compared to 3 millimeters sheath).

Using specific variations in the pattern of grooves, the exact locationof the core stream can be also be moved across the channel. The capacityeither to separate the walls of the channel from the core fluid using aminimum of sheath fluid or to focus the core fluid in a well definedregion within the channel are significant advantages of the sheath flowdevice and method.

Furthermore, the relative flow rates of the core and sheath can bechanged at will and the diameter of the core can be varied in real timeif the application warrants, with no need to alter the device itself. Asshown in the data in Table 1, the sheathing process remains unperturbed,even at sheath/core ratios over 40,000. FIG. 11 a shows a core/sheathratio of 2,100. FIG. 11 b shows a core/sheath ratio of 21,000. FIG. 11 cshows a core/sheath ratio of 42,000. Higher resolution microscopes wouldenable viewing of fluorescence from the core for even smaller corediameters.

TABLE 1 Reynolds Sheath Sheath Core Core Diameter Ratio of Number FlowRate Diameter Flow Rate Calculated Measured Core/Sheath 0.0008 21 mL/min3 mm 10 μL/min   45 microns 75 microns 2,100 0.0008 21 mL/min 3 mm 1μL/min 4.5 microns 25 microns 21,000 0.0016 42 mL/min 3 mm 1 μL/min  3microns 16 microns 42,000

The actual size of the core can be changed relative to the size of thechannel by simply altering the relative flow rates of the core andsheath streams. Furthermore, this change can be effected in real time.Unlike nozzle system traditionally used for flow cytometry or extrusion,there is no need to go to smaller and smaller nozzles which may resultin clogging problems, higher back pressures, and reduced output. Inprevious designs, the core solution must pass through a nozzle or otherconstriction to enter the flow. This presents a potential cloggingpoint, for the solution containing the cells or other particles to beanalyzed. Under the present design, channels can be of uniform size toavoid constrictions and potential clogging points.

Using the device and method described herein, microdialysis could beaccomplished without a membrane. The core stream is recaptured after itis exposed by sheathing to the sheath stream. This exposure provides forthe removal of low molecular weight molecules by diffusion across theinterface of the core stream and the sheath stream. The ability toconduct microdialysis without a membrane prolongs the life of thesystem. Current microdialysis systems operate for limited lifetimes dueto the potential for membrane clogging. Additionally, separations basedon differential solubility as well as differential size can be providedby the device and method described herein. For example, a whole bloodsample could be sheathed into the center of the channel, and allowed toflow for sufficient distance for small molecules to diffuse outward fromthe core into the sheath. Cells and larger molecules such as proteinswill not diffuse as quickly and will tend to stay in the core. The corewould then be unsheathed and recovered, with the smaller moleculesremoved.

The device and method are useful as a means of protecting conduits,including but not limited to, pipes, tubes, ducts, tubing, capillaries,and microfluidic channels, from fouling or corrosion. A thin sheathstream of protective material is formed around the core stream. Thesheath stream need not be the same viscosity as the core stream,therefore a relatively slow moving and thin protective sheath coatingcan be formed to protect the insides of conduits exposed to corrosivecore stream solutions.

The device and methods described herein can also be used to reduce thepower requirement for transporting viscous fluids in conduits, includingbut not limited to, ducts, pipes, tubes, tubing, capillaries, andmicrofluidic channels. Sheathing a viscous fluid in a second fluid oflower viscosity reduces the sheer stress at the conduit wall whichlowers the pressure drop required to generate a given flow rate. Thesheath flow component has been used to generate such a flow, in which acore and a sheath stream of differing viscosity initially enter thedevice side-by-side and the lower viscosity sheath stream sheaths thehigher viscosity core stream.

The relatively low flow resistance of the device means that it can beused to sheath quite high-viscosity systems. This is useful in food andpolymer extrusion applications. The device and method is further usefulin the synthesis of specialty polymeric filaments and tubes. Unlikestandard extrusion technologies, filaments with continuously varyingdiameter can be created. Filaments made in this way can be expected tohave increased elasticity over extruded filaments because of the nativeentropy of the polymer chains. The exact design may also be altered tochange the cross-sectional shape of the resulting polymer strand. Sincethe extrusion device is small, inexpensive, and essentially operates asa passive component, many devices can be fabricated to perform inparallel, such as an array.

The device and methods described are also useful as liquid waveguides.Liquid waveguides have been described for monitoring chemical processesin which light is guided in fluid in a capillary or in the walls of acapillary in order to measure some component of the fluid. The deviceand method can be used for guiding the light in either the core streamor sheath stream for similar measurements, but with the capability formore exact focusing, much greater control of the relative dimensions ofthe light guiding fluid and the other fluid, and the avoidance of walleffects such as scattering of the light from the core by the capillarywall. The capability of guiding light in fluids is particularly usefulin microfluidic systems.

FIG. 12 shows the waveguide application. A chip 31 was fabricated with achannel 14 beginning in the center and spiraling outward to the outlet20 on the outside edge of the chip. A sheath stream inlet 10 and a corestream inlet 12, located near the center of the chip, are in fluidconnection with the channel 14. The fluid transporting structures 22sheathe the core stream within the sheath stream. The sheathed solutionthen travels outward in a spiral of 360 degrees before reaching theoutlet 20. A light source 32 is introduced through a window (not shown)located at the outlet 20.

Core and sheath streams are introduced into the structure at the inlets.The core and sheath streams have approximately equivalent densities. Thecore stream is 70% fructose. The sheath stream is a saturated saltsolution with enough fructose added to match the density of the core.There is a small amount of fluorescent dye in the sheath stream. Thesheath was formed in the center of the chip 31 and then traveled outwardalong an increasing spiral.

FIG. 13 shows the resulting waveguided light 33 when light wasintroduced to the channel from an outlet 20. The light is waveguided 33through a full 360 degrees around the spiral. The light source 32illuminates the higher refractive index stream, which in this case isthe core; however, it could be either the sheath stream or the corestream.

The condition for waveguiding is merely that the core stream and thesheath stream have different refractive indices. The ability tohydrodynamically focus a core down to submicron diameters allows for theproduction of a nearfield optical microscope probe entirely out ofliquid. FIG. 14 shows an example of a nearfield optical microscopeutilizing the present invention. Once the core stream 34 is ensheathedin the sheath stream 36, a tapered nozzle 38 is used to create the taperin the core. The high refractive index core stream 34 is directedthrough the nozzle 38. Light introduced into the core will be waveguideddown to the surface 40. Reflected, scattered, or emitted light will thenbe collected by the waveguide and carried upward for detection. Anotherpossible design may eliminate the need for a nozzle by introducingdielectrophoretic forces to push the core stream out into a fine tip.This design would also be able to use dielectric forces to steer thestream and raster it over the surface. Based on refractive indexmeasurements of the chosen chemistry, the optimal geometry of the tapercan be established. Because a solid tip does not have to be brought intoclose proximity with the surface, this design is well suited for theanalysis of fragile biological samples. It is also well suited toperform liquid-phase photochemistry for nanomachining processes. Thechip is able to raster over a surface using a translation stage.

The device and method of the present invention are useful in particlecounting and flow cytometry. Flow cytometry has proven to be aneffective tool for highly multiplexed screening of environmental samplesin an automated format for continuous monitoring. Systems currently inuse include the Luminex® flow cytometer, which is relatively large andrequires a significant volume of water for sheath fluid—a primary factorlimiting the time of continuous operation to one week. Furthermore, incase of a positive result, a separate aliquot of the sample must beanalyzed for agent confirmation; thus all samples must be divided andtemporarily archived prior to cytometry.

The flow cytometer system of the present invention is useful forcontinuous monitoring for biological warfare contaminants in air orwater. The flow cytometer system is typically provided on a microfluidicchip, and is comprised of a sheath flow device and an interrogationregion. The sheath flow device is used to introduce the core and sheathstreams into a microfluidic channel in such a way that the sheath streamcompletely surrounds the core sample stream, thus preventing fouling ofthe microfluidic channel where the top and bottom of the core samplestream would have touched the channel walls and completely focusing thestream within the interrogation region. Optionally, the sheath and corestream can be separated after the analysis so that each can beseparately recaptured and the sheath fluid reused. The use of themicrofluidic flow cytometer and sheath fluid recapture willsignificantly reduce the footprint of the monitoring system.

The optical interrogation region is comprised of at least one waveguide,which is composed of a photoresist material that is integrated onboardthe microfluidic chip for delivery of excitation light at two differentwavelengths and collection of signal for analysis of 3-colorfluorescence emission and light scatter. Coded Luminex® beads providethe multiplexing capability.

On-chip optical analysis was performed on the core stream using a diodelaser with pigtailed optical fibers to illuminate the core stream. Lightscatter at 90° was measured to detect the passage of yeast cells throughthe illuminated region of the core. Signal tracings, representing thelight scatter signal from five-fold serial dilutions of yeast cells,demonstrated that the light scatter signals were proportional to theconcentration of cells in the flow stream.

The addition of optical elements measuring fluorescence to a flowcytometer on a chip is straightforward using methods and devices knownin the art, such as optical fibers or polymer waveguides and lightsources and filters of the appropriate wavelengths. The types offluorescence analyses using dyes and labeled binding molecules that canbe performed are described extensively in literature using commercialflow cytometers and are well known in the art. In general, the number ofanalytes that can be analyzed simultaneously is a function of the numberof labels that can be excited and discriminated. However, one approachthat enables the performance of highly multiplexed assays relies only onthree-color discrimination. A commercially available version of thisapproach for flow cytometry uses coded beads.

Luminex coded beads are prepared so that two colors of fluorescence areemitted when the bead is excited using a red laser. The ratio of the twocolors indicates the identity of the bead. If target is bound to thebead, that bead can be distinguished from beads with no target bound bythe formation of a complex with another antibody labeled with a thirdfluorescent label (dye, quantum dot, fluorescent nanoparticle) excitedusing a green laser.

Recovery of the particles producing a positive signal is desirable inorder to confirm that the positive result was indeed caused by the toxinor pathogen presumed present according to the results of the screeningimmunoassay. Analysis of a separate aliquot inevitably assumes that thecomposition of the tested and archived sample fractions isidentical—which might be questionable for very low concentration ofagent. Therefore a means for sorting can be included in the microflowcytometer device that can provide the ability to sort particles ofinterest into an on-chip collection chamber to reserve them for furtheranalysis.

The core and sheath streams are first introduced to the same channelfrom a ‘T’ intersection so that they are flowing side-by-side. A seriesof grooves placed in the top and bottom of the channel then serve tocompletely wrap the sheath stream around the core. The sheath fluidbecomes entrained by the grooves and travels above and below the corestream to completely surround it.

Example: A flow cytometer was made using soft lithography in a 3 maskprocess. The channels were assembled from two pieces that were mirrorimages of each other. To produce the template, a 35 micron layer of SU-8was first spun onto a silicon wafer and exposed with the mask definingthe 200 micron wide fluid channels. Then a second 30 microns layer wasspun on and exposed with the second mask, defining the grooves. Finallya third mask was used to expose both layers and define the channels tohold the optical fibers.

When baked and developed, only the exposed portions of the SU-8remained. This created a negative master of one half of the channel. Thenegative masters were then used to cast Sylgard-184 (Dow-Corning) intothe two halves of the chip. When the two halves were aligned and broughttogether, the result was a 70 micron deep fluid channel with 30 microndeep grooves placed in the top and the bottom. The fiber channels were130 micron deep because both layers of SU-8 were exposed. This wassufficient to accommodate the 130 micron OD optical fibers.

The chips have two inlets. The inlet and outlet ports were made in thebottom piece of PDMS with an 18-gauge needle. Fluidic connections to thechips were provided via a PMMA base made using a CNC mill. The base hada set of ports on the face that could be aligned with and pressedagainst ports of the chip. A set of internal channels connected theseports with short lengths of stainless steel tubing along the edge of thebase. These could be used as friction connections to silicone tubing,which was connected to syringe pumps (Cole-Parmer, Vernon Hills, Ill.).

Chip assembly required careful alignment of the two halves. The presenceof the fiber channels assisted with the alignment. As a first step, thebottom piece was placed on the base and the ports on the chip werealigned with the ports on the base. Then the fibers were positioned inthe fiber channels. A single-mode fiber was used for illumination and amultimode fiber was used to collect the scattered light at 90 degrees.The base of each fiber was held in place with tape so that it wouldremain in the channel. A small amount of ethanol was then placed on thebottom piece and the top piece positioned on top. The ethanol preventedthe immediate adhesion of the two pieces of PDMS. The two pieces“clicked” together and were aligned to within about 20 microns, due tothe fibers locking into the fiber channels. A glass slide was thenplaced over the top piece and weak lateral pressures were applied untilthe two pieces were completely aligned. The chip was then left until theethanol evaporated and the adhesion between the two PDMS pieces wasfully developed. A small amount of Sylgard-184® was then applied to theend of each fiber channel and allowed to wick in around the fiber andform a complete seal.

FIG. 15 shows the flow cytometer. The inlets 10, 12 are connected to thechannel 14. The fluid transporting structures 22 wrap the sheath streamaround the core stream, focusing the core stream in the interrogationregion 46. Interrogation, for example, illumination, comes from a singlemode fiber 42. Light was collected by a multimode fiber 44.

On-chip optical analysis was performed on the core stream using a diodelaser with pigtailed optical fibers to illuminate the core stream. Lightscatter at 90° was measured to detect the passage of yeast cells throughthe illuminated region of the core. A series of sample suspensions weremade having concentrations of 222, 41.5, 7.44, 1.66, and 0.313 ppm ofYeast (Fleishmann's active dry) in phosphate buffered saline containing0.01% Tween-20. The sample solutions were introduced into the cytometer.The volumetric flow rates of the sample and the sheath were the same.

FIGS. 16( a)-16(e) show a series of traces of the light scatterresulting from the series in order of increasing concentration. As shownin the signal tracings, representing the light scatter signal fromfive-fold serial dilutions of yeast cells, the light scatter signalswere proportional to the concentration of cells in the flow stream. Thesample core was illuminated with the light from a helium-neon laserintroduced via a single mode optical fiber. Scattered light wascollected at 90 degrees using a multimode fiber and detected with aphotomultiplier tube. FIG. 16 a was a highly diluted sample, showing nocells during the 4-second sampling time. Each successive solution wasroughly 5 times as concentrated as the previous solution. Each of thespikes seen in a plot represents the passage of a cell through theinterrogation region. The number of spikes increases approximately5-fold with the 5-fold increase in concentration.

The device and method of the present invention are also useful for thefabrication of materials. For example, the core stream can contain apolymerizable, condensable, cross-linkable or crystalizable material,which is extruded to the desired diameter using the sheath streaminstead of a solid nozzle or channel. Since the flow cytometer device issmall, inexpensive, and essentially operates as a passive component,many devices can be fabricated to perform in parallel, such as an array.

Materials from which structure can be fabricated include but are notlimited to a wide variety of polymers including polystyrene, butylrubber, polypropylene, polyacrylamide, polysiloxane, andpolymethylmethyacrylate. Biological molecules can be ordered to selfassemble into higher order structures; such molecules could include awide variety of lipids, proteins, carbohydrates and oligonucleotides.Materials that form harder structures could be used including precursorsof glassy materials such as sol gels, as discussed in Sousek et al.,Polymers for Advanced Technologies, 2005, 16:257-261, incorporatedherein in full by reference, or initiators for subsequent deposition ofmetals, calcium, and/or semiconductors. The fluids used can be aqueousor organic. Preferably, the core and sheath fluid are the same phase.

By varying the diameter of the core, tapered materials can befabricated. Nonuniform or tapered geometries for waveguides can begenerated. Controlling the relative rates of sheath and core flow duringpolymerization of filaments provides high precision, tapered structureswith sub-micrometer diameter fluctuations, resulting in uniquewaveguiding properties.

The device and method is further useful in the synthesis of specialtypolymeric filaments and tubes. Unlike standard extrusion technologies,filaments with continuously varying diameter can be created. Filamentsmade in this way can be expected to have increased elasticity overextruded filaments because of the native entropy of the polymer chains.The exact design may also be altered to change the cross-sectional shapeof the resulting polymer strand.

By configuring the grooves or ridges used to transport the sheathstream, non-round shapes can also be obtained. In addition to varyingthe rate of flow to change the diameter of the core, the core fluid canbe pulsed instead of flowed continually to stop and start the corestream to form “particles” or “packets” of core fluid. Once the desiredsize and shape are obtained, the material in the core is polymerized,condensed, cross-linked, or crystallized chemically, optically or byother means known in the art. Due to the geometry of the system, thistype of synthesis can be conducted in continuous manner rather than inbatches. Moreover, the geometry of the system is particularly amenableto the production of high-aspect-ratio structures and filaments that areespecially difficult to produce in quantity.

Shapes that can be fabricated in this method include, but are notlimited to, ovals, ribbons, rods, wires, tubes and filaments. Using thegrooves or ridges on the top and bottom of the channel can bespecifically designed to produce the desired shape. The grooves orridges do not have to be straight but can have a variety ofconfigurations as long as they channel the fluid around the core. Theycan be curved, in the shape of chevrons, angled like “check marks,” orin a variety of other shapes in order to influence the shape of theresultant core fluid. The addition of more inputs and grooves furtherdownstream can be used to expand the repertoire of shapes that can befabricated.

More complex shapes that can be designed and fabricated using grooves orridges include hollow cylinders, filled “sausages,” coated particles,rods with alternating composition, also known as “nano bar codes”.Structures with longitudinal or lateral density or chemical gradientscan be fabricated by introducing gradients into one of the flow streams(longitudinal) or by allowing a reactant to diffuse in or out of thecore while it is in contact with the sheath stream (lateral).

FIG. 17 shows a sheath flow device capable of creating a hollow tubeswithin a hollow tube. A sheath input 10 and a core input 14 areconnected to a channel 14 having a series of fluid transportingstructures 22. A series of successive sheath inputs 10 are provideddownstream towards the outlet 20. Each successive sheath input 10creates a new sheath around all the previously sheathed materials. FIG.18 shows a hollow tube within a hollow tube that was made by using thedevice of FIG. 17 by alternately introducing input streams andensheathing the structures. Core stream 50 is surrounded by successivesheath streams 52, 54, 56 58. Sheath streams 52 and 56 were labeled witha fluorescent dye for contrast.

Typically, the sheath stream is sufficient to move the polymerizedmaterial to the output of the channel. For some materials, however, asthe extruded material polymerizes and its viscosity increases from itsunpolymerized value to infinity, the dynamics of the flow profile withinthe channel may change to the point that feed matching is required tocontrol the fluid velocity and effectively remove the polymerizedmaterial. There are several options available to do feed-matching. In anelastomeric chip, the fluid velocity is controlled by compressing thechannel to cause the fluid to accelerate. Additionally, rollers may beplaced at the exit of the chip so that they impinge on the rod andcontrol the linear exit velocity of the polymerized rod.

Generally, the core contains a polymerizable material and is extruded tothe desired diameter using the sheath stream instead of a solid nozzleor channel. Once the desired shape is obtained, the core material ispolymerized chemically or optically. Due to the geometry of the system,production can be in continuous instead of in batch mode. Moreover, thegeometry of the system is particularly amenable to the production ofhigh aspect ratio structures and filaments which are especiallydifficult to produce in quantity. Since the fabrication device is small,inexpensive, and essentially operates as a passive component, manydevices can be fabricated to perform in parallel, such as an array.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

1. A method for fabricating structures, comprising: providing a channelhaving a proximal end and a distal end, said channel having opposedfacing top and bottom surfaces, said channel having at least one firstfluid transporting structure across said channel located on said topsurface and at least one second fluid transporting structure across saidchannel located on said bottom surface, said first and second fluidtransporting structures being located between said proximal and saiddistal end and on said opposing faces surfaces, facing one anotheracross the channel; and introducing a sheath stream and a core stream atsaid proximal end of said channel, said sheath and core streams flowingdown said channel side by side towards said distal end, wherein saidfluid transporting structures transport said sheath stream across saidtop and bottom surfaces of said channel to surround said core stream,wherein said core stream is a material capable of forming a structure.2. The method of claim 1, further comprising introducing at least asecond sheath stream to create an output of multiple concentric layeredstreams.
 3. The method of claim 1, wherein said structure is tapered. 4.The method of claim 1, wherein said structure is a filament of varyingdiameter.
 5. The method of claim 1, wherein said structure has a shapeselected from the group consisting of ovals, ribbons, rods, wires,tubes, and filaments.